Analytical validation of frataxin proteoforms in blood as biomarkers of Friedreich's ataxia

摘要：作为生物标志物的血液中FRATAXIN蛋白质型的分析验证 弗里德里希共济失调 弗里德赖希共济失调（FA）是一种常染色体隐性遗传疾病，由基因组中的内含子GAA三联体扩增引起。 FXN基因，导致线粒体蛋白共济失调蛋白的表达减少。FA估计影响1 in 50，000人的平均死亡年龄在生命的第四个十年。目前还没有批准的治疗FA的方法， 尽管涉及上调或替换共济失调蛋白的实验方法正在被研究， 测试. Frataxin在血清或血浆中是检测不到的，并且不能使用全血，因为Frataxin是 被认为存在于长寿红细胞中。一种用于分析血小板中共济失调蛋白的测定法，所述血小板具有半- 10天的寿命，这将使治疗干预措施进行测试，是由布莱尔和林奇开发的 labs.该检测是基于稳定同位素稀释免疫纯化二维纳米超高 高效液相色谱/平行反应监测/高分辨质谱（纳米- UPLC-MS/HRMS），并监测来自frataxin的三种胰蛋白酶肽。该测定具有100%的灵敏度， 区分对照组和FA病例的特异性，但常规分析血小板非常 在许多临床环境中具有挑战性。我们现在已经发现，在红细胞中， 具有135个氨基酸（76-210）和N-末端乙酰化的甲硫氨酸残基的共济失调蛋白（同种型E）。它 通过用于共济失调蛋白（1-210）的典型全长形式的另一种剪接位点形式产生。 健康志愿者红细胞中E亚型的含量为26.7 ± 6.4 ng/mL，是健康志愿者的3倍 （n=10）与其他血细胞中存在的成熟线粒体共济失调蛋白（7.1 ± 1.0 ng/mL）相比。 我们最近发现，同种型E（8.5 ± 1.1 ng/mL）和成熟线粒体共济失调蛋白（2.1 ± 1.1 ng/mL） 与健康对照受试者相比，FA患者（n=29）的血液中两者均降低> 70%。 亚型E缺乏线粒体靶向序列，因此它分布于细胞质和细胞核 当在培养的细胞中表达时。特异性定量线粒体外和线粒体内 全血中共济失调蛋白的同种型将使跟踪疾病的进展和监测 治疗干预的有效性。这将需要开发严格验证的检测方法， 解决与使用血液作为生物基质相关的分析前和分析问题， 生物标志物分析，而不是更常见的使用血清或血浆。本提案涉及 这些问题的具体目标如下： 目的1：对成熟共济失调蛋白和异构体E蛋白的收集和储存进行分析前验证 在全血中。目的2：对成熟线粒体定量方法进行分析验证 全血中的共济失调蛋白和共济失调蛋白同种型E。目的3：验证成熟的共济失调蛋白和共济失调蛋白亚型E 来自多个研究中心的对照、FA携带者和FA病例的全血蛋白质型分析。

ABSTRACT: ANALYTICAL VALIDATION OF FRATAXIN PROTEOFORMS IN BLOOD AS BIOMARKERS OF FRIEDREICH’S ATAXIA Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50,000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood could not be used because frataxin was thought to be present in long-lived erythrocytes. An assay for analyzing frataxin in platelets, which have a half- life of 10 days, which would allow therapeutic interventions to be tested, was developed by the Blair and Lynch labs. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (nano- UPLC-MS/HRMS) and is monitoring three tryptic peptides from frataxin. The assay had 100 % sensitivity and specificity for discriminating between controls and FA cases but analyzing platelets on a routine basis is very challenging in many clinical settings. We have now discovered that in erythrocyte is in fact a novel proteoform of frataxin (isoform E) with 135-amino acids (76-210) and an N-terminally acetylated methionine residue. It arises through an alternative splice-site form that is used for the canonical full-length form of frataxin (1-210). There is three times as much isoform E in erythrocytes (26.7 ± 6.4 ng/mL) from the blood of healthy volunteers (n=10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). We recently found that both isoform E (8.5 ± 1.1 ng/mL) and mature mitochondrial frataxin (2.1 ± 1.1 ng/mL) are both reduced by > 70 % in blood from FA patients (n=29) when compared with healthy control subjects. Isoform E lacks a mitochondrial targeting sequence and so it is distributed to both cytosol and the nucleus when expressed in cultured cells. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood would make it possible to follow the progress of the disease and monitor the efficacy of therapeutic interventions. This will require the development of rigorously validated assays that address the pre-analytical and analytical issues that are relevant to the use of blood as a biological matrix for biomarker analysis rather than the more common use of serum or plasma. The present proposal addresses these issues under following specific aims: Aim 1: To conduct pre-analytical validation for collection and storage of mature frataxin and isoform E protein in whole blood. Aim 2: To conduct analytical validation of the method for quantifying mature mitochondrial frataxin and frataxin isoform E in whole blood. Aim 3: Validation of the mature frataxin and frataxin isoform E proteoform analysis in whole blood from controls, FA carriers, and FA cases from multiple sites.